Examinando por Autor "Sepúlveda, Romina V."
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Ítem Interaction between the Linker, Pre-S1, and TRP Domains Determines Folding, Assembly, and Trafficking of TRPV Channels(Cell Press, 2015-08) Garcia-Elias, Anna; Berna-Erro, Alejandro; Rubio-Moscardo, Fanny; Pardo-Pastor, Carlos; Mrkonjić, Sanela; Sepúlveda, Romina V.; Vicente, Rubén; González-Nilo, Fernando; Valverde, Miguel A.Summary Functional transient receptor potential (TRP) channels result from the assembly of four subunits. Here, we show an interaction between the pre-S1, TRP, and the ankyrin repeat domain (ARD)-S1 linker domains of TRPV1 and TRPV4 that is essential for proper channel assembly. Neutralization of TRPV4 pre-S1 K462 resulted in protein retention in the ER, defective glycosylation and trafficking, and unresponsiveness to TRPV4-activating stimuli. Similar results were obtained with the equivalent mutation in TRPV1 pre-S1. Molecular dynamics simulations revealed that TRPV4-K462 generated an alternating hydrogen network with E745 (TRP box) and D425 (pre-S1 linker), and that K462Q mutation affected subunit folding. Consistently, single TRPV4-E745A or TRPV4-D425A mutations moderately affected TRPV4 biogenesis while double TRPV4-D425A/E745A mutation resumed the TRPV4-K462Q phenotype. Thus, the interaction between pre-S1, TRP, and linker domains is mandatory to generate a structural conformation that allows the contacts between adjacent subunits to promote correct assembly and trafficking to the plasma membrane. © 2015 Elsevier Ltd.Ítem The Emergence of New Catalytic Abilities in an Endoxylanase from Family GH10 by Removing an Intrinsically Disordered Region(MDPI, 2022-02-02) Gil-Durán, Carlos; Sepúlveda, Romina V.; Rojas, Maximiliano; Castro-Fernández, Víctor; Guixé, Victoria; Vaca, Inmaculada; Levicán, Gloria; González-Nilo, Fernando D.; Ravanal, María-Cristina; Chávez, RenatoEndoxylanases belonging to family 10 of the glycoside hydrolases (GH10) are versatile in the use of different substrates. Thus, an understanding of the molecular mechanisms underlying substrate specificities could be very useful in the engineering of GH10 endoxylanases for biotechno-logical purposes. Herein, we analyzed XynA, an endoxylanase that contains a (β/α)8-barrel domain and an intrinsically disordered region (IDR) of 29 amino acids at its amino end. Enzyme activity assays revealed that the elimination of the IDR resulted in a mutant enzyme (XynA∆29) in which two new activities emerged: the ability to release xylose from xylan, and the ability to hydrolyze p-nitrophenyl-β-D-xylopyranoside (pNPXyl), a substrate that wild-type enzyme cannot hydrolyze. Circular dichroism and tryptophan fluorescence quenching by acrylamide showed changes in secondary structure and increased flexibility of XynA∆29. Molecular dynamics simulations revealed that the emergence of the pNPXyl-hydrolyzing activity correlated with a dynamic behavior not previously observed in GH10 endoxylanases: a hinge-bending motion of two symmetric regions within the (β/α)8-barrel domain, whose hinge point is the active cleft. The hinge-bending motion is more intense in XynA∆29 than in XynA and promotes the formation of a wider active site that allows the accommodation and hydrolysis of pNPXyl. Our results open new avenues for the study of the relationship between IDRs, dynamics and activity of endoxylanases, and other enzymes containing (β/α)8-barrel domain.Ítem Unveiling Novel Urease Inhibitors for Helicobacter pylori: A Multi-Methodological Approach from Virtual Screening and ADME to Molecular Dynamics Simulations(MDPI, 2024-02-04) Valenzuela-Hormazabal, Paulina; Sepúlveda, Romina V.; Alegría-Arcos, Melissa; Valdés-Muñoz, Elizabeth; Rojas-Pérez, Víctor; González-Bonet, Ileana; Suardíaz, Reynier; Galarza, Christian; Morales, Natalia; Leddermann, Verónica; Castro, Ricardo I.; Benso, BrunaHelicobacter pylori (Hp) infections pose a global health challenge demanding innovative therapeutic strategies by which to eradicate them. Urease, a key Hp virulence factor hydrolyzes urea, facilitating bacterial survival in the acidic gastric environment. In this study, a multi-methodological approach combining pharmacophore- and structure-based virtual screening, molecular dynamics simulations, and MM-GBSA calculations was employed to identify novel inhibitors for Hp urease (HpU). A refined dataset of 8,271,505 small molecules from the ZINC15 database underwent pharmacokinetic and physicochemical filtering, resulting in 16% of compounds for pharmacophore-based virtual screening. Molecular docking simulations were performed in successive stages, utilizing HTVS, SP, and XP algorithms. Subsequent energetic re-scoring with MM-GBSA identified promising candidates interacting with distinct urease variants. Lys219, a residue critical for urea catalysis at the urease binding site, can manifest in two forms, neutral (LYN) or carbamylated (KCX). Notably, the evaluated molecules demonstrated different interaction and energetic patterns in both protein variants. Further evaluation through ADMET predictions highlighted compounds with favorable pharmacological profiles, leading to the identification of 15 candidates. Molecular dynamics simulations revealed comparable structural stability to the control DJM, with candidates 5, 8 and 12 (CA5, CA8, and CA12, respectively) exhibiting the lowest binding free energies. These inhibitors suggest a chelating capacity that is crucial for urease inhibition. The analysis underscores the potential of CA5, CA8, and CA12 as novel HpU inhibitors. Finally, we compare our candidates with the chemical space of urease inhibitors finding physicochemical similarities with potent agents such as thiourea.Ítem β1-subunit-induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel(National Academy of Sciences, 2016-06) Castillo, Juan P.; Sánchez-Rodríguez, Jorge E.; Hyde, H. Clark; Zaelzer, Cristian A.; Aguayo, Daniel; Sepúlveda, Romina V.; Luk, Louis Y.P.; Kent, Stephen B.H.; Gonzalez-Nilo, Fernando D.; Bezanilla, Francisco; Latorre, RamónLarge-conductance Ca2+- and voltage-activated K+ (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit–induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.